Extracellular Matrix-Derived Gels and Related Methods

ABSTRACT

Provided are methods for preparing gelled, solubilized extracellular matrix (ECM) compositions useful as cell growth scaffolds. Also provided are compositions prepared according to the methods as well as uses for the compositions. In one embodiment a device, such as a prosthesis, is provided which comprises an inorganic matrix into which the gelled, solubilized ECM is dispersed to facilitate in-growth of cells into the ECM and thus adaptation and/or attachment of the device to a patient.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119(e) to U.S.Provisional Patent Application No. 60/892,699, filed on Mar. 2, 2007,which is incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERAL FUNDING

This invention was made with government support under Grant No. 5 R01EB000503-04, awarded by the National Institutes of Health. Thegovernment has certain rights in this invention.

Extracellular matrix-derived gels, cell-growth scaffolds and relatedmethods are described herein.

The current trend towards minimally invasive, outpatient-based surgicalprocedures has prompted the development of injectable scaffolds, whichcan be inductive and bioactive or they can be non-inductive “placeholders.” Injectable scaffolds can be used in combination withendoscopic or laparoscopic techniques to deliver bioactive proteinsand/or cells, or bulking agents to target tissues. Purified collagen,gelatin, autologous fat, hyaluronic acid, and synthetic materials areclinically used as injectable scaffolds in regenerative medicine for thetreatment of urinary incontinence, reflux disease, and laryngealpathologies [Lightner D J, et al. Injectable agents: present and future.Curr Urol Rep. 2002 October; 3(5):408-13; Lehman G A. Injectable andbulk-forming agents for enhancing the lower esophageal sphincter. Am J.Med. 2003 Aug. 18; 115 Suppl 3A:188S-91S; Duruisseau O, et al.Endoscopic rehabilitation of vocal cord paralysis with a siliconeelastomer suspension implant. Otolaryngol Head Neck Surg. 2004September; 131(3):241-7]. In addition, purified collagen gels have beeninvestigated in pre-clinical studies as a substrate for the delivery ofneonatal cardiomyocytes to infarcted myocardium [Zhang P, et al.Artificial matrix helps neonatal cardiomyocytes restore injuredmyocardium in rats. Artif Organs. 2006 February; 30(2):86-93] or as aninjectable scaffold for articular surface repair [Xu J W, et al.Injectable tissue-engineered cartilage with different chondrocytesources. Plast Reconstr Surg. 2004 Apr. 15; 113(5):1361-71]. However,overly-purified, chemically modified or synthetic materials can lead toadverse immune responses by the host and limit cell migration into thematrix.

Scaffolds composed of naturally occurring extracellular matrix (ECM)possess many bioactive properties that have been shown to lead toconstructive remodeling of virtually every tissue type with minimizationof scar tissue formation. ECM-derived scaffolds have been used for therepair of a variety of tissues including lower urinary tract structures[Dedecker F, et al. [Small intestinal submucosa (SIS): prospects inurogenital surgery]. Prog Urol. 2005 June; 15(3):405-10; Wood J D,Simmons-Byrd A, et al. Use of a particulate extracellular matrixbioscaffold for treatment of acquired urinary incontinence in dogs. J AmVet Med. Assoc. 2005 Apr. 1; 226(7):1095-7], esophagus [Badylak S, etal. Resorbable bioscaffold for esophageal repair in a dog model. JPediatr Surg. 2000 July; 35(7):1097-103; Badylak S F, et al. Esophagealreconstruction with ECM and muscle tissue in a dog model. J Surg Res.2005 September; 128(1):87-97], cardiac tissue [Badylak S, et al.Extracellular matrix for myocardial repair. Heart Surg Forum. 2003;6(2):E20-6; Badylak S F, et al. The use of extracellular matrix as aninductive scaffold for the partial replacement of functional myocardium.Cell Transplant. 2006; 15 Suppl 1:S29-40; Robinson K A, et al.Extracellular matrix scaffold for cardiac repair. Circulation. 2005 Aug.30; 112(9 Suppl):I135-431, and musculotendinous structures [Badylak S,et al. Naturally occurring extracellular matrix as a scaffold formusculoskeletal repair. Clin Orthop Relat Res. 1999 October(367Suppl):S333-43; Badylak S F, et al. The use of xenogeneic smallintestinal submucosa as a biomaterial for Achilles tendon repair in adog model. J Biomed Mater Res. 1995 August; 29(8):977-85; Zantop T, etal. Extracellular matrix scaffolds are repopulated, in part, by bonemarrow-derived cells in a mouse model of achilles tendon reconstruction.J Orthop Res. 2006 June; 24(6):1299-309] tissues, often leading totissue-specific constructive remodeling without scar formation.

U.S. Pat. No. 5,275,826 discloses an ECM-derived fluidized, injectable,non-immunogenic tissue graft that promotes endogenous tissue growth inthe location of the injection of the tissue graft. The composition iscomprised of tunica submucosa, muscularis mucosa and stratum compactumof the small intestine of a warm-blooded vertebrate.

U.S. Pat. No. 5,516,533 discloses a tissue graft composition comprisedof intestinal submucosa delaminated from both the tunica muscularis (anouter layer of the intestine) and at least the luminal portion of thetunica mucosa (inner layer of the intestine).

U.S. Pat. No. 5,866,414 discloses a cell-growth composition containingprotease-digested submucosal tissue, and added nutrients to support cellgrowth. The submucosal tissue and nutrients are combined in a solution,which is then gelled to form a solid or semi-solid matrix.

U.S. Pat. No. 6,893,666 discloses a composition and methods for using atissue regenerative matrix to promote the restoration, remodeling orrepair of connective tissue. The composition of the matrix comprisesdevitalized mammalian epithelial basement membrane of the intestine andtunica propria, which can further include submucosa, tunica muscularis,growth factors, a cell, or a polymer. The tissue can be obtained fromthe urinary bladder, skin, esophagus and small intestine.

U.S. application Ser. No. 11/182,551 discloses a composition consistingessentially of an emulsified or injectable extracellular matrixcomposition from a mammalian source for regeneration of absent ordefective myocardium. The application also discloses a compositioncomprising synthetic or mammalian extracellular matrix compositions andadditional components, such as a cell, peptide, drug, or nutrient. Theapplication also discloses methods of making and using the composition.Divisional applications related to application Ser. No. 11/182,551include: application Ser. Nos. 11/367,870; 11/448,351; 11/448,355;11/448,931; and 11/448,968. These applications disclose a manner ofpolymerizing the emulsified composition by altering the pH of thecomposition. However, none of the applications discuss the use oftemperature to regulate gelation.

Many forms of ECM scaffolds have already received regulatory approvaland have been used in more than 500,000 human patients. However, thesecurrent forms of ECM are limited by the material and geometricalproperties inherent to the tissue from which they are derived (such assheets or tubes of tissue) and delivery via injection is limited topowder suspensions.

SUMMARY

Provided are methods for preparing gelled, solubilized extracellularmatrix (ECM) compositions useful as cell growth scaffolds. Thecompositions can be molded prior to implantation or administered to apatient in an un-gelled form prior to gelation where the compositiongels in situ. Also provided are compositions prepared according to themethods as well as uses for the compositions. In one embodiment adevice, such as a prosthesis, is provided which comprises an inorganicmatrix into which the gelled, solubilized ECM is dispersed to facilitatein-growth of cells into the ECM and thus adaptation and/or attachment ofthe device to a patient.

In one embodiment, injectable ECM-derived gel scaffolds are providedthat facilitate delivery of the scaffold via minimally invasive methodswhile retaining bioactivity. In another embodiment, the gel can bemolded into any desired shape for use in a patient, such as a humanpatient. The gel scaffold can be used for regenerative or augmentativepurposes, for example to regenerate organs or tissue in a patient, forexample after trauma or surgery to remove tissue, such as a tumor; orfor cosmetic purposes, such as enhancement of facial features or breastreconstruction or augmentation. In one embodiment, the gel scaffold isattached to a biocompatible inorganic matrix, such as, withoutlimitation, a matrix of metal fibers or beads.

According to one embodiment, a method of preparing an extracellularmatrix-derived gel is provided. The method comprising: (i) comminutingan extracellular matrix, (ii) solubilizing intact, non-dialyzed ornon-cross-linked extracellular matrix by digestion with an acid proteasein an acidic solution to produce a digest solution, (iii) raising the pHof the digest solution to a pH between 7.2 and 7.8 to produce aneutralized digest solution, and (iv) gelling the solution at atemperature greater than approximately 25° C. The ECM typically isderived from mammalian tissue, such as, without limitation from one ofurinary bladder, spleen, liver, heart, pancreas, ovary, or smallintestine. In certain embodiments, the ECM is derived from a pig, cow,horse, monkey, or human. In one non-limiting embodiment, the ECM islyophilized and comminuted. The ECM is then solubilized with an acidprotease. The acid protease may be, without limitation, pepsin ortrypsin, and in one embodiment is pepsin. The ECM typically issolubilized at an acid pH suitable or optimal for the protease, such asgreater than about pH 2, or between pH and 4, for example in a 0.01M HClsolution. The solution typically is solubilized for 12-48 hours,depending upon the tissue type (e.g., see examples below), with mixing(stirring, agitation, admixing, blending, rotating, tilting, etc.).

Once the ECM is solubilized (typically substantially completely) the pHis raised to between 7.2 and 7.8, and according to one embodiment, to pH7.4. Bases, such as bases containing hydroxyl ions, including NaOH, canbe used to raise the pH of the solution. Likewise buffers, such as anisotonic buffer, including, without limitation, Phosphate BufferedSaline (PBS), can be used to bring the solution to a target pH, or toaid in maintaining the pH and ionic strength of the gel to targetlevels, such as physiological pH and ionic conditions. The neutralizeddigest solution can be gelled at temperatures approaching 37° C.,typically at any temperature over 25° C., though gelation proceeds muchmore rapidly at temperatures over 30° C., and as the temperatureapproaches physiological temperature. The method typically does notinclude a dialysis step prior to gelation, yielding a more-completeECM-like matrix that typically gels at 37° C. more slowly thancomparable collagen or dialyzed ECM preparations.

As described herein, the composition can be molded into any shape by anysuitable method, including, without limitation, placing into or onto amold, electrospinning, electrodeposition, injection into a cavity oronto a surface in a patient. Further, a molded gel can be trimmed andotherwise shaped by cutting or other suitable methods. In onenon-limiting embodiment, the gel is injected into a site on a patient toadd additional bulk or to fill in a void, for example, resulting fromtrauma or from removal or degradation of tissue. In one non-limitingembodiment, the acidic solubilization solution is mixed in a staticmixer with a base and/or buffer during injection into a patient. Infurther embodiments, cells, drugs, cytokines and/or growth factors canbe added to the gel prior to, during or after gelation, so long as thebioactivity of the cells, drugs, cytokines and/or growth factors is notsubstantially or practically (for the intended use) affected by theprocessing of the gel to its final form.

Also provided is a novel composition prepared according to one or moreprocesses described above or herein, namely, by a method comprising: (i)comminuting an extracellular matrix, (ii) solubilizing intact,non-dialyzed or non-cross-linked extracellular matrix by digestion withan acid protease in an acidic solution to produce a digest solution,(iii) raising the pH of the digest solution to a pH between 7.2 and 7.8to produce a neutralized digest solution, and (iv) gelling the solutionat a temperature greater than 25° C.

In another embodiment a method of preparing a hybrid extracellularmatrix scaffold is provided along with a matrix prepared by that method.A scaffold may be any biocompatible porous, macroporous, microporous,etc. material into which an ECM gel is dispersed or can be dispersed,and which supports the desired bioactivity of the device/scaffold, whichis typically cell growth and/or in-growth. The method comprises coatinga matrix of a biocompatible scaffold with a solubilized extracellularmatrix and gelling the matrix. According to one non-limiting embodiment,the solubilized extracellular matrix can be prepared according to theprocess of: (i) comminuting an extracellular matrix, (ii) solubilizingintact, non-dialyzed or non-cross-linked extracellular matrix bydigestion with an acid protease in an acidic solution to produce adigest solution, and (iii) raising the pH of the digest solution to a pHbetween 7.2 and 7.8 to produce a neutralized digest solution, and theneutralized digest solution is gelled at a temperature greater than 25°C., including variations of this method described above and herein. Inone embodiment, after coating the scaffold, the method further includesultrasonicating the scaffold. According to non-limiting embodiments, thescaffold comprises one or more of a cobalt-chrome alloy, a stainlesssteel, titanium, tantalum, and/or a titanium alloy that optionallycomprises non-metallic and metallic components. In one non-limitingembodiment, the scaffold comprises a commercial pure titanium. Inanother, the scaffold comprises a titanium alloy that comprises one ormore of molybdenum, tantalum, niobium, zirconium, iron, manganese,chromium, cobalt, nickel, aluminum and lanthanum. The titanium alloy maybe an alloy comprising Ti, Al, and V, such as, for example, an alloycomprising about 90% wt. Ti, about 6% wt. Al and about 4% wt. V(Ti6Al4V). In one embodiment, the scaffold comprises filaments. Inanother, fused beads. The scaffold may comprise an inorganic,calcium-containing mineral, such as, without limitation, apatite,hydroxyapatite or a mineral comprising Ca, P and O. The scaffold alsomay comprise a polymer (plastic) and/or a ceramic.

In another embodiment, a biocompatible device is provided. The device iscoated with a hybrid scaffold comprising gelled solubilizedextracellular matrix embedded into a porous scaffold. The device may be,without limitation, a prosthesis or an implant. The prosthesis may be ahand, a forearm, an arm, a foot or a leg prosthesis. In one non-limitingembodiment, the device is a femoral implant for use in a hip-replacementprocedure. The gelled solubilized extracellular matrix is, according toone non-limiting embodiment, prepared by a process comprising: (i)comminuting an extracellular matrix, (ii) solubilizing intact,non-dialyzed or non-cross-linked extracellular matrix by digestion withan acid protease in an acidic solution to produce a digest solution,(iii) raising the pH of the digest solution to a pH between 7.2 and 7.8to produce a neutralized digest solution, and (iv) gelling the solutionat a temperature greater than 25° C. in its variations described aboveand herein.

Lastly, a method of attaching a device to tissue and/or structures of apatient is provided comprising contacting a surface of a devicecomprising a hybrid inorganic/extracellular matrix scaffold comprising agelled solubilized extracellular matrix embedded into a porous inorganicscaffold with a patient's cells for a time period sufficient forin-growth of the patient's cells into the scaffold. The surface may becontacted with the cells and the in-growth occurs in vivo and/or exvivo. Without limitation, the device may be any device, including aprosthesis, as described above or herein. In one embodiment, the gelledsolubilized extracellular matrix is prepared by a process comprising:(i) comminuting an extracellular matrix, (ii) solubilizing intact,non-dialyzed or non-cross-linked extracellular matrix by digestion withan acid protease in an acidic solution to produce a digest solution,(iii) raising the pH of the digest solution to a pH between 7.2 and 7.8to produce a neutralized digest solution, and (iv) gelling the solutionat a temperature greater than 25° C.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows schematically a cross-sectional view of the wall of theurinary bladder (not drawn to scale). The following structures areshown: epithelial cell layer (A), basement membrane (B), tunica propria(C), muscularis mucosa (D), tunica mucosa (E), tunica muscularis externa(F), tunica serosa (G), tunica mucosa (H), and lumen of the bladder (L).

FIG. 2 shows photographs of the porcine urinary bladder matrix (UBM) inits different forms: lyophilized UBM sheet (A), lyophilized UBM powder(B); pepsin-digested solution at a concentration of 10 mg/ml of UBM (C),and gels at 4 mg/ml of UBM and at 8 mg/ml of UBM, where a gel ofcollagen I (Col I) at 4 mg/ml is shown for comparison (D).

FIG. 3 shows results from gel-electrophoresis of UBM and Coll gels.

FIG. 4 shows scanning electron micrograph (SEM) images of UBM gels atdifferent concentrations and at different magnifications: 3 mg/ml UBMgel at 5,000×(A) and at 10,000×(B); and 6 mg/ml UBM gel at 5,000×(C) andat 10,000×(D).

FIG. 5 shows SEM images of a 4 mg/ml Col I gel and of a 4 mg/ml UBM gelsat a magnification of 5,000×.

FIG. 6 shows turbidimetric gelation kinetics of Col I gels and UBM gels,which was determined spectrophotometrically by measuring absorbanceduring gelation. Results are shown for both measured absorbance values(A) and normalized absorbance values (B), which allows for calculatingkinetic parameters such as t1/2 (time to reach 50% of maximumturbidity), tlag (lag time of gelation) and S (speed of gelation).

FIG. 7 shows turbidimetric gelation kinetics of 1 mg/mL small intestinesubmucosa (SIS) gels.

FIG. 8 shows rheological measurements during the gelation of UBM gels,where gelation was determined mechanically by monitoring the oscillatorymoduli of the sample at a fixed frequency during gelation. Results areshown of the elastic modulus (G′) and of the viscosity modulus (G″) for3 mg/ml UBM gel and for 6 mg/ml UBM gel.

FIG. 9 shows rheological measurements during the gelation of LS (liverstroma) and SIS gels. Gelation kinetics was determined at 5% strain and1 rad/sec. where results are shown of the elastic modulus (G′) for LS,SIS and UBM gels at 6 mg/mL (A). The storage modulus (G′) as a functionof frequency was also determined for LS, UBM and SIS gels at 6 mg/ml(B).

FIG. 10 shows the effect of frequency on the dynamic viscosity of 3mg/ml Coll gel, 3 mg/ml UBM gel and 6 mg/ml UBM gel (A).

FIG. 11 shows the results of an adhesion assay with rat aortic smoothmuscle cells (rSMCs) in culture after 30 minutes, where rSMCs werecultured on Col I gel, UBM gel (UBM-g) and lyophilized UBM sheets(UBM-Lyo). Results are shown for adhesion of rSMCs relative to adhesionof rSMCs on tissue culture plastic (TCP), where the activity of attachedcells was determined by a MTT assay (n=3).

FIG. 12 shows the results of a MTT assay to determine the viability ofrSMC cultures after 7 days on either Col I gel or on UBM gel (UBM-g)(n=4, *p<0.05).

FIG. 13 shows histological images of rSMC cultures after 7 days. Imagesare shown for rSMCs (shown by arrows) at 10× on the abluminal (A) andluminal (B) sides of the UBM-Lyo sheets, where samples were fixed andstained with H & E. Images are also shown for cultures at 10× (C) and at20× (D) on UBM gels, where samples were fixed and stained with Masson'strichrome.

FIG. 14 shows the results of a MTT assay to determine the viability ofrSMC cultures after 3 hours and after 48 hours on different substrates:tissue culture plate (TCP), Col I (collagen type I gel), UBM (Urinarybladder matrix gel), LS (porcine liver stroma gel), spleen (spleen ECMgel), UBM-Lyo (lyophilized UBM sheet) and UBM-Hy (hydrated UBM sheets).All gels were at 6 mg/ml.

FIG. 15 shows the results of a chemotaxis assay of human aorticendothelial cells (HAECs) for different solutions: dilutions of digestsolution of UBM with a solution containing acid and pepsin (UBM),dilutions of EBM-2 media with a solution containing acid and pepsin(Buffer), endothelial basal cell medium (EBM-2) and fetal bovine serum(FBS). Chemotaxis was assessed using CytoSelect™ 96-well Cell MigrationAssay, where relative fluorescence units (RFU) correlated to the numberof migratory cells that achieved chemotaxis.

FIG. 16 shows the results of a MTT assay to determine the viability ofhuman microvascular endothelial cells on different substrates: tissueculture plate (TCP), Collagen (collagen type I gel), UBM (Urinarybladder matrix gel), LS (porcine liver stroma gel), and spleen (spleenECM gel). All gels were at 6 mg/ml and cells were seeded in triplicates(n=3).

FIG. 17 shows digital photographs of a porous titanium fiber penetratedwith UBM gel (stained in turquoise), where the fiber was treated without(A) and with (B) ultrasonication. Scale bars are 2000 μm.

FIG. 18 shows SEM images of porous metal scaffolds and ESEM(environmental scanning electron microscopy) images of hybridextracellular matrix (ECM)/porous metal scaffolds. SEM images are shownof a porous metal scaffold containing Ti6Al4V wires in a fiber mesh (A)or containing sintered commercially pure titanium (CP Ti) beads (C).ESEM images are shown of the hybrid ECM/porous metal scaffold, where UBMgel coats both the Ti6Al4V wires (B) and the CP Ti beads (D) afterexposure to ultrasonication.

FIG. 19 shows schematically one embodiment of a femoral implantdescribed herein.

FIG. 20 shows schematically one embodiment of a hand prosthesisdescribed herein.

DETAILED DESCRIPTION

Methods are described herein of preparing an injectable and bioactiveextracellular matrix (ECM)-derived composition comprising solubilizedextracellular matrix obtained from any of a variety of tissues. Relatedcompositions, devices and methods of use also are described. Theviscosity of the matrix increases when warmed to physiologicaltemperatures approaching about 37° C. According to one non-limitingembodiment, the ECM-derived composition is an injectable solution attemperatures lower than 37° C., but a gel at a physiological temperatureof 37° C. According to certain embodiments, the gel is bioactive becausethe entire, intact ECM is solubilized and is not dialyzed, cross-linkedand/or otherwise treated to remove or otherwise inactivate ECMstructural or functional components, resulting in a highly bioactive gelscaffold that is functionally superior to earlier-described matrices. Ageneral set of principles for preparing an ECM-derived gel is providedalong with specific protocols for preparing gels from numerous tissues,including urinary bladder, spleen, liver, heart, pancreas, ovary andsmall intestine.

The compositions described herein find use as, without limitation, aninjectable graft (e.g., xenogeneic, allogeneic or autologous) fortissues, for example, bone or soft tissues, in need of repair oraugmentation most typically to correct trauma or disease-induced tissuedefects. The compositions also may be used as a filler for implantconstructs comprising, for example, a molded construct formed into adesired shape for use in cosmetic or trauma-treating surgicalprocedures.

The compositions may be implanted into a patient, human or animal, by anumber of methods. In one non-limiting embodiment, the compositions areinjected as a liquid into a desired site in the patient. The compositionmay be pre-seeded with cells, and then preferably injected using alarger-bore, e.g. 16 gauge needle, to prevent shearing of cells. Inanother non-limiting embodiment, the composition is gelled within amold, and the gelled, molded product is then implanted into the patientat a desired site. The gelled, molded product may be pre-seeded (laidonto the molded gel or mixed in during gelation) with cells, such ascells of the patient.

As used herein, the terms “extracellular matrix” and “ECM” refer to anatural or artificial scaffolding for cell growth. Natural ECMs (ECMsfound in multicellular organisms, such as mammals and humans) arecomplex mixtures of structural and non-structural biomolecules,including, but not limited to, collagens, elastins, laminins,glycosaminoglycans, proteoglycans, antimicrobials, chemoattractants,cytokines, and growth factors. In mammals, ECM often comprises about 90%collagen, in its various forms. The composition and structure of ECMsvary depending on the source of the tissue. For example, small intestinesubmucosa (SIS), urinary bladder matrix (UBM) and liver stroma ECM eachdiffer in their overall structure and composition due to the uniquecellular niche needed for each tissue.

As used herein, the terms “intact extracellular matrix” and “intact ECM”refers to an extracellular matrix that retains activity of itsstructural and non-structural biomolecules, including, but not limitedto, collagens, elastins, laminins, glycosaminoglycans, proteoglycans,antimicrobials, chemoattractants, cytokines, and growth factors, suchas, without limitation comminuted ECM as described herein. The activityof the biomolecules within the ECM can be removed chemically ormechanically, for example, by cross-linking and/or by dialyzing the ECM.Intact ECM essentially has not been cross-linked and/or dialyzed,meaning that the ECM has not been subjected to a dialysis and/or across-linking process, or conditions other than processes that occurnaturally during storage and handling of ECM prior to solubilization, asdescribed herein. Thus, ECM that is substantially cross-linked and/ordialyzed (in anything but a trivial manner which does not substantiallyaffect the gelation and functional characteristics of the ECM in itsuses described herein) is not considered to be “intact”.

By “Bio Compatible”, it is meant that a device, scaffold composition,etc. is essentially, practically (for its intended use) and/orsubstantially non-toxic, non-injurous or non-inhibiting ornon-inhibitory to cells, tissues, organs, and/or organ systems thatwould come into contact with the device, scaffold, composition, etc.

In general, the method of preparing an ECM-derived gel requires theisolation of ECM from an animal of interest and from a tissue or organof interest. In certain embodiments, the ECM is isolated from mammaliantissue. As used herein, the term “mammalian tissue” refers to tissuederived from a mammal, wherein tissue comprises any cellular componentof an animal. For example and without limitation, tissue can be derivedfrom aggregates of cells, an organ, portions of an organ, combinationsof organs. In certain embodiments, the ECM is isolated from a vertebrateanimal, for example and without limitation, human, monkey, pig, cattle,and sheep. In certain embodiments, the ECM is isolated from any tissueof an animal, for example and without limitation, urinary bladder,liver, small intestine, esophagus, pancreas, dermis, and heart. In oneembodiment, the ECM is derived from a urinary bladder. The ECM may ormay not include the basement membrane portion of the ECM. In certainembodiments, the ECM includes at least a portion of the basementmembrane. The ECM may or may not retain some of the cellular elementsthat comprised the original tissue such as capillary endothelial cellsor fibrocytes.

As used herein, the term “derive” and any other word forms of cognatesthereof, such as, without limitation, “derived” and “derives”, refers toa component or components obtained from any stated source by any usefulmethod. For example and without limitation, an ECM-derived gel refers toa gel comprised of components of ECM obtained from any tissue by anynumber of methods known in the art for isolating ECM. In anotherexample, mammalian tissue-derived ECM refers to ECM comprised ofcomponents of mammalian tissue obtained from a mammal by any usefulmethod.

The ECM can be sterilized by any number of standard techniques,including, but not limited to, exposure to peracetic acid, low dosegamma radiation, gas plasma sterilization, ethylene oxide treatment orelectron beam treatment. More typically, sterilization of ECM isobtained by soaking in 0.1% (v/v) peracetic acid, 4% (v/v) ethanol, and95.9% (v/v) sterile water for two hours. The peracetic acid residue isremoved by washing twice for 15 minutes with PBS (pH=7.4) and twice for15 minutes with sterile water.

Following isolation of the tissue of interest, decellularization isperformed by various methods, for example and without limitation,exposure to hypertonic saline, peracetic acid, Triton-X or otherdetergents. Sterilization and decellularization can be simultaneous. Forexample and without limitation, sterilization with peracetic acid,described above, also can serve to decellularize the ECM. DecellularizedECM can then be dried, either lyophilized (freeze-dried) or air dried.Dried ECM can be comminuted by methods including, but not limited to,tearing, milling, cutting, grinding, and shearing. The comminuted ECMcan also be further processed into a powdered form by methods, forexample and without limitation, such as grinding or milling in a frozenor freeze-dried state.

As used herein, the term “comminute” and any other word forms orcognates thereof, such as, without limitation, “comminution” and“comminuting”, refers to the process of reducing larger particles intosmaller particles, including, without limitation, by grinding, blending,shredding, slicing, milling, cutting, shredding. ECM can be comminutedwhile in any form, including, but not limited to, hydrated forms,frozen, air-dried, lyophilized, powdered, sheet-form.

In order to prepare solubilized ECM tissue, comminuted ECM is digestedwith an acid protease in an acidic solution to form a digest solution.As used herein, the term “acid protease” refers to an enzyme thatcleaves peptide bonds, wherein the enzyme has increased activity ofcleaving peptide bonds in an acidic pH. For example and withoutlimitation, acid proteases can include pepsin and trypsin.

The digest solution of ECM typically is kept at a constant stir for acertain amount of time at room temperature. The ECM digest can be usedimmediately or be stored at −20° C. or frozen at, for example andwithout limitation, −20° C. or −80° C. To form a “pre-gel” solution, thepH of the digest solution is raised to a pH between 7.2 and 7.8. The pHcan be raised by adding one or more of a base or an isotonic bufferedsolution, for example and without limitation, NaOH or PBS at pH 7.4. Themethod typically does not include a dialysis step prior to gelation,yielding a more-complete ECM-like matrix that typically gels at 37° C.more slowly than comparable collagen or dialyzed ECM preparations. Thegel is therefore is more amenable to injection into a patient, and alsoretains more of the qualities of native ECM due to retention of manynative soluble factors, such as, without limitation, cytokines. Theability of non-dialyzed (whole ECM) preparations prepared from a varietyof tissues to gel with kinetics suitable for use in molds or in situ isunexpected.

As used herein, the term “isotonic buffered solution” refers to asolution that is buffered to a pH between 7.2 and 7.8 and that has abalanced concentration of salts to promote an isotonic environment. Asused herein, the term “base” refers to any compound or a solution of acompound with a pH greater than 7. For example and without limitation,the base is an alkaline hydroxide or an aqueous solution of an alkalinehydroxide. In certain embodiments, the base is NaOH or NaOH in PBS.

This “pre-gel” solution can, at that point be incubated at a suitablywarm temperature, for example and without limitation, at about 37° C. togel. The pre-gel can be frozen and stored at, for example and withoutlimitation, −20° C. or −80° C. As used herein, the term “pre-gelsolution” or “pre-gel” refers to a digest solution wherein the pH isincreased. For example and without limitation, a pre-gel has a pHbetween 7.2 and 7.8.

Any type of extracellular matrix tissue can be used in the methods,compositions and devices as described herein (see generally, U.S. Pat.Nos. 4,902,508; 4,956,178; 5,281,422; 5,352,463; 5,372,821; 5,554,389;5,573,784; 5,645,860; 5,771,969; 5,753,267; 5,762,966; 5,866,414;6,099,567; 6,485,723; 6,576,265; 6,579,538; 6,696,270; 6,783,776;6,793,939; 6,849,273; 6,852,339; 6,861,074; 6,887,495; 6,890,562;6,890,563; 6,890,564; and 6,893,666). In certain embodiments, the ECM isisolated from a vertebrate animal, for example and without limitation,from a warm blooded mammalian vertebrate animal including, but notlimited to, human, monkey, pig, cow and sheep. The ECM can be derivedfrom any organ or tissue, including without limitation, urinary bladder,intestine, liver, esophagus and dermis. In one embodiment, the ECM isisolated from a urinary bladder. The ECM may or may not include thebasement membrane portion of the ECM. In certain embodiments, the ECMincludes at least a portion of the basement membrane.

In another embodiment, the ECM is prepared by abrading porcine bladdertissue to remove the outer layers including both the tunica serosa andthe tunica muscularis (layers G and F in FIG. 1) using a longitudinalwiping motion with a scalpel handle and moistened gauze. Followingeversion of the tissue segment, the luminal portion of the tunica mucosa(layer H in FIG. 1) is delaminated from the underlying tissue using thesame wiping motion. Care is taken to prevent perforation of thesubmucosa (layer E of FIG. 1). After these tissues are removed, theresulting ECM consists mainly of the tunica submucosa (layer E of FIG.1).

The ECM can be sterilized by any of a number of standard methods withoutloss of its ability to induce endogenous tissue growth. For example, thematerial can be sterilized by propylene oxide or ethylene oxidetreatment, gamma irradiation treatment (0.05 to 4 mRad), gas plasmasterilization, peracetic acid sterilization, or electron beam treatment.The material can also be sterilized by treatment with glutaraldehyde,which causes cross linking of the protein material, but this treatmentsubstantially alters the material such that it is slowly resorbed or notresorbed at all and incites a different type of host remodeling whichmore closely resembles scar tissue formation or encapsulation ratherthan constructive remodeling. Cross-linking of the protein material canalso be induced with carbodiimide or dehydrothermal or photooxidationmethods. More typically, ECM is disinfected by immersion in 0.1% (v/v)peracetic acid (a), 4% (v/v) ethanol, and 96% (v/v) sterile water for 2h. The ECM material is then washed twice for 15 min with PBS (pH=7.4)and twice for 15 min with deionized water.

Commercially available ECM preparations can also be used in the methods,devices and compositions described herein. In one embodiment, the ECM isderived from small intestinal submucosa or SIS. Commercially availablepreparations include, but are not limited to, Surgisis™, Surgisis-ES™,Stratasis™, and Stratasis-ES™ (Cook Urological Inc.; Indianapolis, Ind.)and GraftPatch™ (Organogenesis Inc.; Canton Mass.). In anotherembodiment, the ECM is derived from dermis. Commercially availablepreparations include, but are not limited to Pelvicol™ (sold asPermacol™ in Europe; Bard, Covington, Ga.), Repliform™ (Microvasive;Boston, Mass.) and Alloderm™ (LifeCell; Branchburg, N.J.). In anotherembodiment, the ECM is derived from urinary bladder. Commerciallyavailable preparations include, but are not limited to UBM (AcellCorporation; Jessup, Md.).

Tissue for preparation of ECM can be harvested in a large variety ofways and once harvested, a variety of portions of the harvested tissuemay be used. For example and without limitation, in one embodiment, theECM is isolated from harvested porcine urinary bladder to prepareurinary bladder matrix (UBM). Excess connective tissue and residualurine are removed from the urinary bladder. The tunica serosa, tunicamuscularis externa, tunica submucosa and most of the muscularis mucosa(layers G, F, E and mostly D in FIG. 1) can be removed mechanicalabrasion or by a combination of enzymatic treatment, hydration, andabrasion. Mechanical removal of these tissues can be accomplished byabrasion using a longitudinal wiping motion to remove the outer layers(particularly the abluminal smooth muscle layers) and even the luminalportions of the tunica mucosa (epithelial layers). Mechanical removal ofthese tissues is accomplished by removal of mesenteric tissues with, forexample, Adson-Brown forceps and Metzenbaum scissors and wiping away thetunica muscularis and tunica submucosa using a longitudinal wipingmotion with a scalpel handle or other rigid object wrapped in moistenedgauze. The epithelial cells of the tunica mucosa (layer A of FIG. 1) canalso be dissociated by soaking the tissue in a de-epithelializingsolution, for example and without limitation, hypertonic saline. Theresulting UBM comprises basement membrane of the tunica mucosa and theadjacent tunica propria (layers B and C of FIG. 1), which is furthertreated with peracetic acid, lyophilized and powdered. Additionalexamples are provided below and are also present in the related art.

In another embodiment, the epithelial cells can be delaminated first byfirst soaking the tissue in a de-epithelializing solution such ashypertonic saline, for example and without limitation, 1.0 N saline, forperiods of time ranging from 10 minutes to 4 hours. Exposure tohypertonic saline solution effectively removes the epithelial cells fromthe underlying basement membrane. The tissue remaining after the initialdelamination procedure includes epithelial basement membrane and thetissue layers abluminal to the epithelial basement membrane. This tissueis next subjected to further treatment to remove the majority ofabluminal tissues but not the epithelial basement membrane. The outerserosal, adventitial, smooth muscle tissues, tunica submucosa and mostof the muscularis mucosa are removed from the remainingde-epithelialized tissue by mechanical abrasion or by a combination ofenzymatic treatment, hydration, and abrasion.

In one embodiment, the ECM is prepared by abrading porcine bladdertissue to remove the outer layers including both the tunica serosa andthe tunica muscularis (layers G and F in FIG. 1) using a longitudinalwiping motion with a scalpel handle and moistened gauze. Followingeversion of the tissue segment, the luminal portion of the tunica mucosa(layer H in FIG. 1) is delaminated from the underlying tissue using thesame wiping motion. Care is taken to prevent perforation of thesubmucosa (layer E of FIG. 1). After these tissues are removed, theresulting ECM consists mainly of the tunica submucosa (layer E of FIG.1).

The compositions described herein can be used in a number of ways orforms. For example and without limitation, according to a firstembodiment, the pre-gel is placed in a suitable mold to model an organor a portion thereof. As a non-limiting example, the composition ismolded into a portion of a liver to facilitate re-growth of livertissue. In another non-limiting example, the composition is molded inthe shape of nose or ear cartilage, or a portion thereof, forreplacement of damaged or excised cartilaginous tissue. In yet anothernon-limiting example, the composition is molded into the shape of awound to facilitate non-scarring healing of that tissue. To prepare themolded gel, the pre-gel is placed in a biocompatible and preferablysterile mold, such as a plastic mold, and is incubated at a temperatureand for a time suitable for gelation of the composition, for example andwithout limitation at about 37° C. In one embodiment, the compositionand mold is placed in an incubator at 37° C. to gel. Because CO₂ hasbeen found to slow gelation, in one non-limiting embodiment, CO₂ is notinjected into the incubator, though in yet another embodiment, CO₂and/or temperature may be used to control the gelation process.

Any useful cytokine, chemoattractant or cells can be mixed into thecomposition prior to gelation or diffused, absorbed and/or adsorbed bythe gel after it is gelled. For example and without limitation, usefulcomponents include growth factors, interferons, interleukins,chemokines, monokines, hormones, angiogenic factors, drugs andantibiotics. Cells can be mixed into the neutralized solubilized gel orcan be placed atop the molded composition once it is gelled. In eithercase, when the gel is seeded with cells, the cells can be grown and/oradapted to the niche created by the molded ECM gel by incubation in asuitable medium in a bioreactor or incubator for a suitable time periodto optimally/favorably prepare the composition for implantation in apatient. The molded composition can be seeded with cells to facilitatein-growth, differentiation and/or adaptation of the cells. For exampleand without limitation, the cells can be autologous or allogeneic withrespect to the patient to receive the composition/device comprising thegel. The cells can be stem cells or other progenitor cells, ordifferentiated cells. In one example, a layer of dermis obtained fromthe patient is seeded on a mold, for use in repairing damaged skinand/or underlying tissue.

As used herein, the terms “mold” refers to a cavity or surface used toform the gel into a three-dimensional shape. For example and withoutlimitation, the mold can be a well plate, cell culture dish or a tube orcan be shaped into any useful shape. In a certain embodiment, the moldcan be shaped into a certain organ or part of an organ. The gel can bedelivered to the mold in a variety of methods, including, but notlimited to, injection, deposition.

As used herein, the terms “drug” and “drugs” refer to any compositionshaving a preventative or therapeutic effect, including and withoutlimitation, antibiotics, peptides, hormones, organic molecules,vitamins, supplements, factors, proteins and chemoattractants.

As used herein, the terms “cell” and “cells” refer to any types of cellsfrom any animal, such as, without limitation, rat, mice, monkey, andhuman. For example and without limitation, cells can be progenitorcells, such as stem cells, or differentiated cells, such as endothelialcells, smooth muscle cells. In certain embodiments, cells for medicalprocedures can be obtained from the patient for autologous procedures orfrom other donors for allogeneic procedures.

One favorable aspect of the use of pre-molded tissue is that a layeredcomposition can be produced. For example, a core portion of thecomposition to be implanted can be prepared with a first ECM gel,obtained from a first source, and a surrounding layer can be with asecond ECM gel, obtained from a second source different from the first,or the same source as the first, but containing different constituents,such as cytokines or cells.

In another embodiment of the pre-molded composition, the ECM gel iscontained within a laminar sheath of non-comminuted and non-digested ECMtissue, such as SIS or UBM, to add physical strength to the gel. In thisembodiment, sheets of ECM tissue, prepared in any manner known in theart, can be placed into the mold prior to filling the mold with thesolubilized ECM tissue for producing the gel. The sheets of ECM tissuemay be used as the mold, so long as they are formed and sewn orcross-linked into a desired shape. In this manner, a solid compositioncan be produced that has greater physical strength than is the case of agel, alone.

In another non-limiting embodiment, the composition is injected as apre-gel into a patient. The composition is injected at a locus in thepatient where the matrix is needed for cell growth. For example andwithout limitation, where a patient has had tissue removed due totrauma, debridement and/or removal of damaged, diseased or canceroustissue, the composition can be injected at the site of tissue removal tofacilitate in-growth of tissue. The viscosity of the pre-gel can becontrolled by varying the amounts of water (e.g., by varying the amountsof water, acid, base, buffer (such as PBS) or other diluents) used toprepare the pre-gel. In applications in which a small gauge needle isused, such as in endoscopy, a less viscous pre-gel would be needed,which typically results in a less viscous gel, once the pre-gel isgelled. In applications in which a larger gauge needle is available, amore viscous gel, with higher strength when gelled, can be used. Also,use of a larger gauge needle, irrespective of the viscosity of thepre-gel, favors mixing of live cells with the pre-gel immediately priorto implantation with less risk of shearing the cells.

In one embodiment, a pre-gel is prepared by raising the pH of the acidicdigest solution and the pre-gel is directly injected into a patientprior to significant gelation proceeds. In one embodiment, the pre-gelis in a frozen state and is thawed and warmed prior to injection. Inanother embodiment, the acidic digest solution is warmed tophysiological temperature and is mixed during injection in a staticmixer with suitable quantities of a base and/or buffer, such as PBS.Suitable static mixers include, without limitation, helical or squarestatic mixers, commercially available from Cammda Corporation ofCobourg, Ontario, Canada or a Mini-Dual Syringe with Micro Static Mixercommercially available from Plas-Pak Industries, Inc. of Norwich, Conn.

In a further embodiment, a commercial kit is provided comprising acomposition described herein. A kit comprises suitable packagingmaterial and the composition. In one non-limiting embodiment, the kitcomprises a pre-gel in a vessel, which may be the packaging, or whichmay be contained within packaging. In this embodiment, the pre-geltypically is frozen or kept at near-freezing temperatures, such as,without limitation, below about 4° C. In another non-limitingembodiment, the kit comprises a first vessel containing an acidicsolution comprising digest solution of ECM as described herein, and asecond vessel comprising a neutralizing solution comprising a baseand/or buffer(s) to bring the acidic solution of the first vessel tophysiological ionic strength and pH, to form a pre-gel. This kit alsooptionally comprises a mixing needle and/or a cold-pack. The vessel maybe a vial, syringe, tube or any other container suitable for storage andtransfer in commercial distribution routes of the kit.

In yet another embodiment of the kit, a pre-gel composition is moldedand pre-gelled prior to packaging and distribution. In one embodiment,the molded gel is packaged in a blister-pack comprising a plasticcontainer and a paper, plastic and/or foil sealing portion, as arewell-known in the art. The mold and packaging typically is sterilizedprior to or after packaging, for example and without limitation, bygamma irradiation. The molded composition may be packaged in anysuitable physiological solution, such as PBS or saline. If the moldedgel contains live cells, the mold can be transported in a suitablecell-culture medium in a sealed jar or other vessel. Of course, thecell-containing molded gel would have to be shipped in an expeditedmanner to preserve the cells.

As used herein, the term “hybrid inorganic/ECM scaffold” refers to aECM-derived gel that is coated onto a biocompatible inorganic structure,such as, without limitation, a metal, an inorganic calcium compound suchas calcium hydroxide, calcium phosphate or calcium carbonate, or aceramic composition. In one embodiment, ultrasonication is used to aidin coating of the inorganic structure with the ECM-derived gel. As usedherein, the term “ultrasonication” refers to the process of exposingultrasonic waves with a frequency higher than 15 kHz and lower than 400kHz.

As used herein, the term “coat”, and related cognates such as “coated”and “coating,” refers to a process comprising of covering an inorganicstructure with ECM-derived gel or hybrid inorganic/ECM scaffold. Forexample and without limitation, coating of an inorganic structure withECM-derived gel can include methods such as pouring, embedding,layering, dipping, spraying.

In another embodiment of the technology described herein, thecomposition is coated onto a biocompatible structural material, such asa metal, an inorganic calcium compound such as calcium hydroxide,calcium phosphate or calcium carbonate, or a ceramic composition.Non-limiting examples of suitable metals are cobalt-chrome alloys,stainless steel alloys, titanium alloys, tantalum alloys,titanium-tantalum alloys, which can include both non-metallic andmetallic components, such as molybdenum, tantalum, niobium, zirconium,iron, manganese, chromium, cobalt, nickel aluminum and lanthanum,including without limitation, CP Ti (commercially pure titanium) ofvarious grades or Ti 6Al 4V (90% wt. Ti, 6% wt. Al and 4% wt. V),stainless steel 316, Nitinol (Nickel-titanium alloy), titanium alloyscoated with hydroxyapatite. Metals are useful due to high strength,flexibility, and biocompatibility. Metals also can be formed intocomplex shapes and many can withstand corrosion in the biologicalenvironments, reduce wear, and not cause damage to tissues. In onenon-limiting example, the metal is femoral or acetabular component usedfor hip repair. In another example, the metal is a fiber or otherprotuberance used in permanent attachment of a prosthesis to a patient.Other compositions, including ceramics, calcium compounds, such as,without limitation, aragonite, may be preferred, for example and withoutlimitation, in repair of or re-shaping of skeletal or dental structures.Combinations of metal, ceramics and/or other materials also may proveuseful. For instance, a metal femoral component of a hip replacement maycomprise a ceramic ball and/or may comprise a plastic coating on theball surface, as might an acetabular component.

Metals, as well as other materials, as is appropriate, can be useful inits different forms, including but not limited to wires, foils, beads,rods and powders, including nanocrystalline powder. The composition andsurface of metals or other materials can also be altered to ensurebiocompatibility, such as surface passivation through silane treatments,coating with biocompatible plastics or ceramics, composite metal/ceramicmaterials. The materials and methods for their employment are well-knownin the field of the present invention.

A difficulty with using metal inserts to repair a patient's skeletalstructure is that the inserts must be anchored/attached to existingskeletal parts. Traditional methods employed cement and/or screws. Inthe case of prostheses, the prostheses are not connected to a patient'stissue except, typically, by cementing. Therefore, it is desirable tobiologically attach a patient's tissue to a medical device. This may beaccomplished by coating surfaces of the implant with the ECM geldescribed herein, which will facilitate in-growth of tissue and thusattachment of the device. A variety of porous structures can be attachedto the implant to create a scaffold into which the ECM gel, and latercells or other tissue (e.g., bone) can infiltrate. Structures include,without limitation: woven or non-woven mesh, sponge-like porousmaterials, fused beads, etc. The porous scaffold will facilitateformation of a strong bond between living tissue, including bone, andthe device. The “pores” of the porous scaffold may be of any size thatwill permit infiltration of an ECM gel, optionally facilitated byultrasound or other treatments that would assist in permeation of thegel, and later cells or other biological materials, such as bone,cartilage, tendons, ligaments, fascia or other connective tissue, intothe scaffolding. In one embodiment, metal fibers are attached to thedevice, and the metal fibers are coated with an ECM gel describedherein, thereby permitting in-growth of tissue within the fibers. In asecond embodiment, a matrix of small beads is welded or otherwiseattached to a surface of the device and an ECM gel described herein iscoated onto the bead matrix, facilitating in-growth of tissue among thebeads. In one example, a device contains a protuberance of fibers, whichcan be inserted inside a bone, permitting fusion of the metal fiberswith the bone. In one embodiment, the ECM gel is seeded and incubatedwith a suitable cell population, such as autologous osteoblasts, tofacilitate bone in-growth.

In another embodiment, the hybrid inorganic/ECM scaffold can also beused to coat other structural implants, such as, without limitation, afemoral implant, a prosthesis of the hand. FIG. 19 shows schematicallyone embodiment of a device 10 inserted into a femur 15 in a hipreplacement procedure. FIG. 19 illustrates device 10, showing an insertportion 20 for insertion into femur 15, and an extension 25 into which aball (not shown) is screwed or otherwise inserted. Device 10 comprises aporous coating 30 of, for example and without limitation, metal beadswelded onto the device 10. Region A in FIG. 19 shows a magnified view ofcoating 30 of device 10. Beads 32 are welded to metal surface 34 ofdevice 10. ECM gel 36 is coated onto and between beads 32. Bone tissuegrowth into beads 32 is facilitated by the presence of the ECM gel 36.

A prosthesis might be anchored into bone in a like manner using aninsert having a porous coating, with the porous coating extending to thelimits of where attachment to a patient's tissue is desired. As anexample, shown in FIG. 20, a hand prosthesis 100 comprises an externalportion 115 and an internal portion 120, which comprises a radius insertportion 122 and an ulnar insert portion 124. Porous coating 130 extendsfrom insert portions 122 and 124 for attachment to bone, to thebeginning of external portion 115, permitting attachment of dermis andintermediary tissue between the bones and dermis.

In use, the device which is coated with a suitable scaffolding and ECMgel as described herein may be contacted with cells, e.g. of a patientor allogeneic cells, and the cells are allowed to infiltrate the matrix.The in-growth or infiltration of cells can occur in vivo or ex vivo,depending on optimization of methods. For example and withoutlimitation, in the case of a femoral implant, the implant can beinserted into the femur and cells of a patient, and desirable bonetissue, infiltrates the scaffolding to fuse the device to the bone. Inanother embodiment, for example in the case of an artificial tendon orligament, a biopsy of a patient's tendons or ligaments is incubated withan appropriate scaffold in order to create an autologous ligament ortendon graft.

EXAMPLES Example 1 Preparation of Porcine Extracellular Matrix (ECM)(UBM)

The preparation of UBM has been previously described [Sarikaya A, et al.Tissue Eng. 2002 February; 8(1):63-71; Ringel R L, et al. J Speech LangHear Res. 2006 February; 49(1):194-208]. In brief, porcine urinarybladders were harvested from 6-month-old 108-118 kg pigs(Whiteshire-Hamroc, IN) immediately following euthanasia. Connectivetissue and adipose tissue were removed from the serosal surface and anyresidual urine was removed by repeated washes with tap water. The tunicaserosa, tunica muscularis externa, the tunica submucosa, and majority ofthe tunica muscularis mucosa were mechanically removed. The urothelialcells of the tunica mucosa were dissociated from the luminal surface bysoaking the tissue in 1.0 N saline solution yielding a biomaterialcomposed of the basement membrane plus the subjacent tunica propria,which is referred to as urinary bladder matrix (UBM).

The UBM sheets were disinfected for two hours on a shaker in a solutioncontaining 0.1% (v/v) peracetic acid, 4% (v/v) ethanol, and 95.9% (v/v)sterile water. The peracetic acid residue was removed by washing withsterile phosphate-buffered saline (pH=7.4) twice for 15 minutes each andtwice for 15 minutes each with sterile water. The UBM sheets (as in FIG.2A) were then lyophilized (FIG. 2B) using a FTS Systems Bulk FreezeDryer Model 8-54 and powdered using a Wiley Mini Mill.

One gram of lyophilized UBM powder (FIG. 2B) and 100 mg of pepsin wereboth mixed in 100 ml of 0.01 M HCl. The solution was kept at a constantstir for ˜48 hrs at room temperature (25° C.). After pepsin digestion,the digest solution (FIG. 1C) was aliquoted and stored at −20° C. untiluse. After completion, the solution is referred to as digest solution orECM digest or ECM stock solution.

Example 2 Preparation of Porcine Spleen ECM

Fresh spleen tissue was obtained. Outer layers of the spleen membranewere removed by slicing, where remaining tissue was cut into uniformpieces. Remnants of outer membrane were trimmed, then rinsed three timesin water. Water was strained by using a sieve. Splenocytes were lysed bymassaging. Spleen slices were incubated in a solution of 0.02%trypsin/0.05% EDTA at 37° C. for 1 hour in a water bath. If necessary,splenocytes were further lysed by massaging. After rinsing, slices weresoaked in 3% Triton X-100 solution and put on a shaker for 1 hour. Ifnecessary, splenocytes were further lysed by massaging. Slices were thensoaked in 4% deoxycholic acid solution and put on a shaker for 1 hour.After thoroughly rinsing, the purified spleen ECM was stored for furtherprocessing. This tissue was next disinfected with peracetic acidtreatment and dried.

One gram of dry porcine spleen ECM and 100 mg of pepsin were both mixedin 100 ml of 0.01 M HCl. The solution was kept at a constant stir for˜72 hrs at room temperature (25° C.). If there are no visible pieces ofthe ECM floating in the solution, aliquot the sample and freeze (−20°C.) or use immediately.

Example 3 Preparation of Porcine Liver Stroma ECM

Fresh liver tissue was obtained. Excess fat and tissue were trimmed.Outer layers of the liver membrane were removed by slicing, whereremaining tissue was cut into uniform pieces. Remnants of outer membranewere trimmed using a scalpel or razor blade, then rinsed three times inwater. Water was strained by using a sieve. Cells were lysed bymassaging. Liver slices were incubated in a solution of 0.02%trypsin/0.05% EDTA at 37° C. for 1 hour in a water bath. If necessary,cells were further lysed by massaging. After rinsing, slices were soakedin 3% Triton X-100 solution and put on a shaker for 1 hour. Ifnecessary, cells were further lysed by massaging. Slices were thensoaked in 4% deoxycholic acid solution and put on a shaker for 1 hour.After thoroughly rinsing, the purified liver stroma was stored indeionized water for further processing. This tissue was next disinfectedwith peracetic acid treatment and dried.

One gram of dry porcine liver stroma ECM and 100 mg of pepsin were bothmixed in 100 ml of 0.01 M HCl. The solution was kept at a constant stirfor ˜24-48 hrs at room temperature (25° C.). If there are no visiblepieces of the ECM floating in the solution, aliquot the sample andfreeze (−20° C.) or use immediately.

Example 4 Preparation of Human Liver Stroma ECM

Fresh liver tissue was obtained. Excess fat and tissue were trimmed.Outer layers of the liver membrane were removed by slicing, whereremaining tissue was cut into uniform pieces. Remnants of outer membranewere trimmed using a scalpel or razor blade, then rinsed three times inwater. Water was strained by using a sieve. Cells were lysed bymassaging. Liver slices were incubated in a solution of 0.02%trypsin/0.05% EDTA at 37° C. for 1 hour in a water bath. If necessary,cells were further lysed by massaging. After rinsing, slices were soakedin 3% Triton X-100 solution and put on a shaker for 1 hour. Ifnecessary, cells were further lysed by massaging. Slices were thensoaked in 4% deoxycholic acid solution and put on a shaker for 1 hour.After thoroughly rinsing, the purified liver stroma was stored indeionized water for further processing. This tissue was next disinfectedwith peracetic acid treatment and dried.

One gram of dry human liver stroma ECM and 200 mg of pepsin were bothmixed in 50 ml of 0.01 M HCl. The solution was kept at a constant stirfor ˜3-5 days at room temperature (25° C.). The solution will need to bemonitored every day. If there are no visible pieces of the ECM floatingin the solution, aliquot the sample and freeze (−20° C.) or useimmediately.

Example 5 Preparation of Porcine Cardiac ECM

One gram of dry porcine cardiac ECM with 100 mg of pepsin were bothmixed in 50 mL of 0.01 M HCl. The solution was kept at a constant stirfor ˜48 hours at room temperature (25° C.).

Example 6 Preparation of Porcine Pancreatic ECM

One gram of dry de-fatted porcine pancreatic ECM with 100 mg of pepsinwere both mixed in 50 mL of 0.01 M HCl. The solution was kept at aconstant stir for ˜48 hours at room temperature (25° C.).

Example 7 Preparation of Porcine Ovarian ECM

Fresh ovarian tissue is obtained within 6 hours of harvest. Ovaries wereremoved and stored in physiological saline tissue until ready fordissection and residual uterine tissue was removed. Longitudinalincisions were made through the hilum of the ovary and the follicleswere disrupted. Once all the follicles have been disrupted, the ECM hasbeen harvested from the ovaries. Rinse three times in filtered water andstrain the water using a sieve. Cells were lysed by gentle massaging.ECM was incubated in a solution of 0.02% trypsin/0.05% EDTA at 37° C.for 1 hour in a water bath and then rinsed. If necessary, cells werefurther lysed by massaging. ECM was soaked in 3% Triton X-100 solutionand put on a shaker for 1 hour. After rinsing, cells were further lysedby massaging if necessary. Slices were then soaked in 4% deoxycholicacid solution and put on a shaker for 1 hour. After thoroughly rinsingto remove residual surfactant, the ECM was stored in sterile/filteredwater until further use. This tissue was next disinfected with peraceticacid treatment and dried.

One gram of lyophilized ovarian ECM powder and 100 mg of pepsin wereboth mixed in 100 ml of 0.01 M HCl. The solution was kept at a constantstir for ˜48 hrs at room temperature (25° C.). After pepsin digestion,the digest solution was aliquoted and stored at −20° C. until use.

Example 8 General Method of Preparation of Gels from ECM

UBM gel was formed into a gel by mixing 0.1 N NaOH ( 1/10 of the volumeof digest solution) and 10×PBS pH 7.4 ( 1/9 of the volume of digestsolution) in appropriate amounts at 4° C. The solution was brought tothe desired volume and concentration using cold (4° C.) 1×PBS pH 7.4 andplaced in a 37° C. incubator for gelation to occur (FIG. 2D).

The ECM was able to form a matrix after 40 minutes in solution as shownin FIG. 2. The ECM-derived gel was liquid at temperatures below 20° C.but turn into a gel when the temperature is raised to 37° C.

In preparing gels from ECM, all of the following solutions should bekept on ice and the following variables must be determined:

-   -   C_(f)=concentration of the final gel in mg/ml    -   C_(s)=concentration of the ECM digest solution in mg/ml    -   V_(f)=volume of the final gel solution needed for the        experiments    -   V_(d)=volume needed from the ECM digest solution in ml    -   V_(10×)=volume of 10×PBS needed in ml    -   V_(1×)=volume of 1×PBS needed in ml    -   V_(NaOH)=volume of 0.1 N NaOH needed in ml

First, determine the final concentration (C_(f)) and volume (V_(f)) ofECM gel required. Then, calculate the mass of ECM needed by multiplyingC_(f) (mg/ml)*V_(f) (ml). This value will give you the volume neededfrom the ECM digest solution (V_(d)), where V_(d)=[C_(f) (mg/ml)*V_(f)(ml)]/C_(s).

Calculate the volume of 10×PBS needed by dividing the calculated volumeV_(d) by 9 (V_(10×)=V_(d)/9). Calculate the volume of 0.1 N NaOH neededby dividing the calculated volume V_(d) by 10 (V_(NaOH)=V_(d)/10).Calculate the amount of 1×PBS needed to bring the solution to theappropriate concentration/volume as follow:V_(1×)=V_(f)−V_(d)−V_(10×)−V_(NaOH). Add all the reagents(V_(1×)+V_(d)+V_(10×)+V_(NaOH)) to an appropriate container (usually 15or 50 ml centrifuge tubes) without the ECM digest (V_(d)). Placesolutions on ice and keep on ice at all times.

Add the appropriate volume from the ECM digest solution (V_(d)) to thePBS/NaOH mixture prepared above and mix well with a 1 ml micropipettewhile being careful and avoiding the creation of air bubbles in thesolution. Depending on the viscosity of the ECM digest solution, theremight be some significant volume loss during the transfer. Monitor thetotal volume and add appropriate amounts until the final volume isachieved. Measure the pH of the pre-gel solution, where pH should bearound 7.4.

Add the pre-gel solution to a mold or to appropriate wells. Place themold or wells in 37° C. incubator for a minimum of 40 minutes. Avoidusing an incubator with CO₂ control. If water evaporation is a concern,place the mold inside a plastic zip-lock bag before placing in theincubator. After gelation, the gel can be removed from the mold andplaced on 1×PBS. If the gels were made in tissue culture plates, 1×PBScan be placed on top of the gels until use to maintain the gelshydrated.

Sample calculation: Make 6 ml of gel with a final concentration of 6mg/ml from the 10 mg/ml stock solution.

GIVEN: C _(s)=10 mg/ml; C _(f)=6 mg/ml; V _(f)=6 ml

V _(d)=[6 mg/ml*6 ml]/10 mg/ml=3.600 ml

V _(10×)=3.6/9=0.400 ml

V _(NaOH)=3.6/10=0.360 ml

V _(1×)=6 ml−3.6 ml−0.400 ml−0.360=1.640 ml

Example 9 Composition and Morphology of Porcine UBM

UBM and rat-tail collagen type I (BD, Biosciences) solutions wereelectrophoresed on 4-20% polyacrylamide gels under reducing conditions(5% 2-Mercaptoethanol). The proteins were visualized with Gel-Code Blue(Bio-Rad), and documented by a Kodak imaging station.

Collagen and sulfated glycosaminoglycan (S-GAG) content were determinedusing the hydroxyproline assay [Reddy G K, Enwemeka C S. A simplifiedmethod for the analysis of hydroxyproline in biological tissues. ClinBiochem. 1996 June; 29(3):225-9] and the Blyscan™ assay kit (Biocolor,Northern Ireland) respectively (three samples were tested). The Blyscan™assay was performed according to the manufacturer's instruction. Thehydroxyproline content was determined by hydrolyzing the samples with 2M NaOH (100 μl total volume) in an autoclave at 120° C. for 20 minutes.The samples were neutralized with 50 μl of 4 M HCl and reacted with 300μl of 0.056 M chloramine-T (Spectrum), mixed gently, and allowed tooxidize for 25 minutes at room temperature. The samples were then mixedwith 300 μl of 1 M Ehrlich's aldehyde (Spectrum) and incubated at 65° C.for 20 minutes. A standard curve was generated using rat-tail collagentype I (BD Biosciences) and used to calculate the total amount ofcollagen present in the digested UBM solutions. The colorimetric changewas determined by the absorbance at 550 nm using a SpectraMaxspectrophotometer.

The composition of the gel has been determined. The collagenconcentration for pepsin digested UBM was found to be 0.8±0.2 mg per mgof dry lyophilized UBM powder (mean±SD). The total S-GAG content wasfound to be 5.1±0.9 μg per mg of dry lyophilized UBM powder (mean±SD).The electrophoresed proteins show the typical bands for collagen type Ipresent on the UBM lane with extra bands as shown in FIG. 3. Thedifference may be in part due to the additional components, that is, tosmall peptides and glycosaminoglycans) present in the UBM gels.

The surface morphology of the UBM gels was examined using a scanningelectron microscope (SEM). The specimens were fixed in cold 2.5%glutaraldehyde and rinsed in PBS, followed by a dehydration processthrough a graded series of ethanol (30% to 100%), and finally criticallypoint dried in an Emscope CPD 750 critical point dryer. The samples wereattached to aluminum SEM specimen mounting stubs (Electron MicroscopySciences, Hatfield, Pa.) and sputter coated with a gold palladium alloyusing a Sputter Coater 108 Auto (Cressington Scientific Instruments,Valencia, Pa.). Finally, samples were examined using a scanning electronmicroscope (JEOL 6330F). Images were taken at a 5,000 and 10,000×magnification. The scanning electron microscopy pictures show thefibrillar appearance of the UBM gels at concentrations of 3 mg/ml and 6mg/ml (FIG. 4A-4D) as well as at 4 mg/ml (FIG. 5B).

Example 10 Rheological Properties and Gelation Kinetics of Porcine UBM,SIS and LS Gels

The rheological properties of the UBM derived gel was characterizedduring gelation. The UBM gel consists of a viscous solution attemperature below 25° C. and a gel at physiological temperatures (37°C.). Rheological properties of other gels can be measured using similarmethods described herein. Rheological properties of liver stroma (LS)and small intestine submucosa (SIS) were also measured.

Turbidimetric gelation kinetics was determined spectrophotometrically aspreviously described [Gelman R A, et al. Collagen fibril formation.Evidence for a multistep process. J Biol. Chem. 1979 Jan. 10;254(1):180-6]. Final pre-gel solutions at the appropriate concentrationwere kept at 4° C. and transferred to a cold 96 well plate by placing100 μl per well in triplicates. The SpectraMax spectrophotometer(Molecular Devices) was pre-heated to 37° C., the plate was placed inthe spectrophotometer, and the turbidity of each well was measured at405 nm every 2 minutes for 1.5 hours (FIG. 6A). Turbidity can also bemeasured at 530 nm (FIG. 7). The absorbance values were recorded andnormalized as shown in FIG. 6B. The time needed to reach 50% of themaximum turbidity measurement (e.g. maximum absorbance value) wasdefined as t_(1/2) and the lag phase (t_(lag)) was calculated byextrapolating the linear growth of the curve. The speed (S) of thegelation based on turbidimetric measurements was determined bycalculating the slope of the growth portion of the curve as shown inFIG. 6B.

Dynamic oscillatory measurements are commonly used in fundamentalstudies of gelation and in characterizing the viscoelastic properties ofgels. The sample was subjected to an oscillatory strain of:

γ(t)=γ₀ cos(2πft)  (1)

where γ₀ was the amplitude of the sinusoidal strain, t was the time, andf was the frequency. The sample developed a sinusoidal stress describedas follow:

σ(t)=|G*|γ(t)  (2)

where G* was the frequency dependent complex modulus of the sample. Thereal part of G*, denoted G′, was in phase with the applied strain andwas called the storage modulus since it corresponded to storage ofmechanical energy in the elastic deformation of the sample. Theimaginary portion of G*, denoted G″, was 90° out of phase with theapplied strain and was called the loss modulus since it corresponded tothe loss of energy by viscous dissipation within the sample. Since thesample was expected to develop solid-like characteristics as gelationproceeds, G′ was expected to increase significantly.

A final property of interest was the magnitude of the complex viscositydefined as follows:

$\begin{matrix}{{\eta^{*}} = {\frac{G^{*}}{2\pi \; f} = \frac{\sqrt{G^{\prime 2} + G^{''2}}}{2\pi \; f}}} & (3)\end{matrix}$

where |η*| was the frequency dependent complex viscosity, G* was thefrequency dependent complex modulus, and f was the frequency. It iscommon to fit complex viscosity versus frequency data to a power-law ofthe form:

|η*|=kf ^(n)  (4)

where k and n are both constants.

Rheological experiments were performed with a TA Instruments AR2000stress-controlled rheometer using a 40 mm-diameter parallel plategeometry and a Peltier cell to maintain the sample temperature. Thesamples were prepared as discussed earlier and loaded into the rheometerwith the Peltier cell maintaining a temperature of 15° C. The sampleedge was protected from evaporation by applying mineral oil. Theviscosity of the sample was first measured by applying a constant stressof 1 Pa on the sample for one minute at 15° C. The temperature was thenset to 37° C. to induce gelation; the Peltier cell typically reached atemperature of 30° C. within 10 seconds and 37° C. within 50 seconds.During this increase in temperature and the subsequent gelation, theoscillatory moduli of the sample were monitored continuously at a fixedfrequency of 0.159 Hz (1 rad/s) and a strain of 5%. When there was nofurther change in the elastic modulus (G′) with time, gelation wasdeemed to be complete. The final linear viscoelastic properties of thegel were measured by performing a frequency sweep between 15.9 Hz and0.08 Hz at 37° C. and 5% strain and fitted to equation 4.

The turbidimetric gelation kinetics and the calculated parameters areshown in FIG. 6 and the results presented in Table 1. The turbidimetricgelation kinetics for UBM and collagen type I gels followed a sigmoidalshape (FIG. 6A). Collagen type I gels at a concentration of 3 mg/mlbecame more turbid following gelation than UBM-gel at a concentration of3 mg/ml and 6 mg/ml (FIG. 6A). The lag phase (t_(lag)) and the timerequired to reach half the final turbidity (t_(1/2)) were greater in theUBM gel (at 3 and 6 mg/ml) than collagen type I (3 mg/ml). In addition,the speed of the turbidimetric gelation kinetics (5) was lower for UBMwhen compared to collagen type I. There was no change in t_(lag),t_(1/2), and S in UBM gels with a change in concentration but there wasa change in the maximum turbidity reached.

Turbidimetric kinetics of 1 mg/mL SIS gel also followed a sigmoidalshape (FIG. 7). Whereas UBM measurements were obtained at 405 nm, SISmeasurements were obtained at 530 nm. SIS measurements also displayed adecrease in turbidity before maximum turbidity was reached.

Both the storage modulus (G′) and the loss modulus (G″) of UBM gelschanged over time with a sigmoidal shape after the temperature of thesample was raised from 15° C. to 37° C. (FIG. 8). G′ and G″ reached asteady state after approximately 8 minutes, suggesting that gelation hadoccurred. The kinetics of G′ and G″ were faster than the turbidimetrickinetics. The viscosities of both UBM and collagen type I are shown inFIG. 10 over a frequency range of ˜0.08-15 Hz and the results aresummarized in Table 1.

The storage modulus (G′) of LS and SIS gels also changed over time witha sigmoidal shape after the temperature of the sample was raised to 37°C. (FIG. 9A). Kinetics of G′ for both LS and SIS gels were faster thankinetics of G′ for UBM gels. The storage modulus (G′) of LS, SIS and UBMgels were also measured as a function of angular frequency (FIG. 9B).

TABLE 1 Results from the turbidimetric analysis of the UBM gelationkinetics. Data represents mean ± SD. Three samples were tested (n = 3).Material k t_(1/2) t_(lag) Collagen type I 3 mg/ml  0.20 (0.01)*  12.2(1.1)*  9.7 (0.8)* UBM 3 mg/ml 0.07 (0.01) 24.4 (2.4) 15.8 (2.0) UBM 6mg/ml 0.09 (0.04) 22.4 (4.9) 14.1 (3.7) *p < 0.05

In an effort to explore the feasibility of using UBM as an injectablematerial, multiple trials were performed to test them in an injectionsetting. ECM powder suspended in saline and UBM gels were tested side byside to see if they could successfully pass through injection needlesfrequently used in medical procedures such as vocal cord augmentation.These needles had 1 cm long, 25 gauge caliber tips that are attached to25 cm long, 16 gauge needle shafts. UBM gels easily and consistentlypassed through these needles. The UBM powder suspension had an upperlimit concentration of 10 mg/ml above which the needle would befrequently occluded, making it difficult to determine the actual amountof ECM delivered. This trial showed the feasibility of using the UMB gelas an injectable material (Table 2).

TABLE 2 Comparison of the viscosity of UBM gels with injectablematerials commercially available. Frequency Range Material k n r² [Hz]REF Urinary 2.35 −1.0617 0.988 0.01-15 — Bladder Matrix 3 mg/ml Urinary5.69 −0.9547 0.999 0.01-15 — Bladder Matrix 6 mg/ml Gelatin 149.39−0.9030 0.997 0.01-15 Chan et al. ^(a) (Gelfoam) Zyplast ™ 99.851−0.9145 0.998 0.01-15 Chan et al. ^(a) Zyderm ™ 66.395 −0.9154 0.9980.01-15 Chan et al. ^(a) Zyderm ™ 12 −0.860 0.977  0.01-100 Klemuk etal. ^(b) Cymetra ® 19.9 −0.778 0.972  0.01-100 Klemuk et al. ^(b)Hyaluronic 3.19 −0.744 0.974  0.01-100 Klemuk et al. ^(b) Acid-DTPHHuman ab- 23.576 −0.9508 0.994 0.01-15 Chan et al. ^(a) dominal sub-cutaneous fat Polytetraflu- 1151.9 −1.0267 0.997 0.01-15 Chan et al.^(a) oroethylene (PTFE) ^(a) Chan R W, et al. Viscosities of implantablebiomaterials in vocal fold augmentation surgery. Laryngoscope. 1998 May;108(5): 725-31. ^(b) Klemuk S A, et al. Viscoelastic properties of threevocal-fold injectable biomaterials at low audio frequencies.Laryngoscope. 2004 September; 114(9): 1597-603.

Example 11 Adhesion and Proliferation assays with Rat Smooth MuscleCells (rSMCs)

The preparation of UBM has been previously described [Freytes, D O etal, Biomaterials, 2004. 25(12): 2353-61]. Briefly, porcine urinarybladders were harvested and the tunica serosa, tunica muscularisexterna, tunica submucosa, and most of the tunica muscularis mucosa weremechanically removed. The resulting biomaterial was composed of thebasement membrane plus the subjacent tunica propria. This bi-laminatestructure was referred to as urinary bladder matrix or UBM. UBM sheetswere disinfected for two hours in a 0.1% (v/v) peracetic acid solution.UBM sheets were either lyophilized or lyophilized and powdered afterprocessing.

One gram of lyophilized UBM powder and 100 mg of pepsin were mixed in100 mL of 0.01 M HCl and kept at a constant stir for ˜48 hrs at roomtemperature (25° C.). UBM and rat tail collagen type I gels were made bybringing the pH and the ionic strength to physiological range using1×PBS in a 37° C. incubator. Gel formation kinetics was determined bymeasuring the absorbance (570 nm) every 2 minutes for ˜1.5 hrs. Gelswere properly fixed and imaged using scanning electron microscopy (SEM).Equal amounts of each solution were electrophoresed on a gradient 4-20%polyacrylamide gel under reducing conditions (5% 2-Mercaptoethanol). Theproteins were visualized with Gel-Code Blue (Bio-Rad), and documented bya Kodak imaging station. Collagen and sulfated glycosaminoglycan (S-GAG)content were determined using the hydroxyproline assay and the Blyscan™assay kit (Biocolor, Northern Ireland).

Rat smooth muscle cells (rSMCs) were harvested as previously described[Ray J L, Leach R, Herbert J M, Benson M. Isolation of vascular smoothmuscle cells from a single murine aorta. Methods Cell Sci. 2001;23(4):185-8] and expanded in DMEM with low bicarbonate and supplementedwith 10% fetal bovine serum (FBS) and 100 U/ml Penicillin/100 μg/mlStreptomycin. The adhesion and proliferation of rSMCs was measured byseeding the surface of 6 mm disks of collagen type I and UBM gels intriplicates. The disks were prepared by adding 100 μl of the appropriategel (3 mg/ml) onto wells of 96 well plates.

Adhesion experiments were performed with rSMCs suspended in serum freeDMEM and seeded at a concentration of 4×10⁴ per well for 30 minutes.Non-adherent cells were removed and the activity of the attached cellswas quantified using the MTT assay. The MTT assay is a colorimetric testthat measures cell viability by activity of mitochondria within thecells, where increased absorbance at 570 nm relates to increasedactivity of enzymes within the mitochondria. rSMCs showed similaradhesion on collagen type I (Col I), UBM gels, and lyophilized UBMsheets (FIG. 11).

Proliferation experiments were performed with rSMCs at three differentconcentrations (1, 2 and 4×10⁴ cells per well), and the activity of thecells was determined using the MTT assay by following the manufacturer'sinstructions. rSMCs successfully adhered to UBM gels and were able togrow for 48 hours with a slight increase in cell activity when comparedto cells grown on collagen type I (Table 3). rSMCs cultured for one weekalso showed an increase in mitochondrial activity on UBM gels whencompared to the activity on collagen type I gels (see FIG. 12).

TABLE 3 Results form the proliferation and adhesion assays using primaryrat aortic endothelial cells. Data represents mean ± SD. Three sampleswere tested (n = 3). % of Cellular Activity when Assay Initial CellNumber Compared to Collagen Type I 48 hrs 1 × 10⁴ 124 (12) proliferation2 × 10⁴ 111 (13) 4 × 10⁴ 119 (7)  30 min adhesion 4 × 10⁴  73 (29)

Growth of rSMCs on collagen type I gels, UBM gels, and UBM lyophilizedsheets was also examined histologically. Disks of UBM gel were madeusing a stainless steel ring (1.5 cm in diameter) as a mold. rSMCs wereseeded on the top surface of the gel at a density of 0.5×10⁶ cells/cm².Media was changed every other day and the cells were allowed to grow for10 days. The samples where then fixed with 10% buffered formalin andstained using H&E or Masson's Trichrome stain. rSMCs formed a confluentmultilayer after the 7-10 day incubation period on both UBM gels and UBMlyophilized sheets (FIG. 13). Contraction of the UBM and collagen type Igels was observed which could suggest a change of the rSMCs from aproliferative state to a contractile state when seeded on the gels,which shows that cell-matrix interactions and traction forces wereformed during in vitro culture

Cell viability was also measured over a period of 48 hours on differenttypes of ECM-derived gels. rSMCs were seeded at 0.125×10⁶ cells/cm² intriplicates on the surface of different substrates and the MTT assayused to determine cell viability at 3 and 48 hours following seeding(FIG. 14). TCP=Tissue Culture Plate; Col I=Purified Collagen Type I Gel,UBM=Urinary Bladder Matrix Gel; LS=Porcine Liver Stroma Gel;Spleen=Spleen ECM Gel; UBM-Lyo=Lyophilized UBM sheet; andUBM-Hy=Hydrated UBM sheets. All gels were at a 6 mg/ml concentration.

Preliminary implantation of 6 mg/ml UBM gels on a subcutaneous pocket ofa rat showed complete degradation of the scaffold after 14 days with nosigns of inflammation (unpublished results). Together, these data showthe potential cytocompatibility of the UBM gels but further in vivotesting is required.

Example 12 Chemotaxis assay with Human Aortic Endothelial Cells

The solubilized UBM also retains its bioactivity such as chemoattractantproperties, where human aortic endothelial cells (HAECs) migratedtowards a UBM digest solution more than towards a solution containingpepsin alone (FIG. 15, comparing data from “Buffer 1:10” to “UBM 1:10”).

Chemotaxis assay was assessed using CytoSelect™ 96-well Cell MigrationAssay following the manufacturer's instructions. Briefly, a membranewith a small pore size discriminates between migratory and non-migratorycells. Migratory cells extend protrusions towards the chemoattractantson the other side of the membrane and pass through the pores. Thesemigratory cells are dissociated from the membrane and detected byfluorescence with CyQuant® GR Dye (Invitrogen), which binds to cellularnucleic acid. Therefore, increased relative fluorescence units (RFU)correlated to a higher number of migratory cells that achievedchemotaxis through the membrane.

Example 13 Adhesion and Proliferation Assay with Human AorticEndothelial Cells

Human microvascular endothelial cells (at the mentioned Initial CellNumber per well) were seeded in triplicates on the surface of differentsubstrates and the MTT assay used to determine cell viability (FIG. 16).TCP=Tissue Culture Plate; Collagen=Purified Collagen Type I, UBM=UrinaryBladder Matrix Gel; LS=Porcine Liver Stroma Gel; Spleen=Spleen ECM Gel.All gels were at a 6 mg/ml concentration.

Example 14 Hybrid Inorganic/ECM Scaffold

Restoration of joint kinematics after limb amputation and replacementwith a prosthesis is limited due to the inability to attach existingmusculature to the prosthesis via boney insertion of tendons [Higuera,C. A., et al.: J Orthop Res. 1091-9 (23) 2005]. Although a variety ofporous titanium and tantalum alloys have been successful at promotingbone ingrowth, there are no alternatives to promote the ingrowth offibrocartilaginous tissue that restores a ligament or tendon insertionsite. Recently, porous tantalum scaffolds have been investigated fortheir ability to promote ingrowth of a vascularized fibrous tissue withpromising mechanical strength [Hacking, S. A., et al.: J Biomed MaterRes, 631-8 (52) 2000]. Naturally derived extracellular matrix (ECM)scaffolds from the porcine small intestine and urinary bladder (UBM)have also been shown to form well organized tendon, ligament, cartilage,and bone, as well as strong boney insertion sites with good mechanicalstrength [Badylak, S. F.: Transpl Immunol. 367-77 (12) 2004; Dejardin,L. M., et al.: AJSM. 175-84 (29) 2001]. It is reasonable to expect thata porous tantalum or titanium scaffold with an ECM embedded within thepores may improve the ingrowth of soft tissue into the metal surface andpromote the formation of fibrocartilaginous tissue. The goal of thecurrent study was to determine the feasibility of coating a poroustitanium scaffold with an ECM gel for the eventual application ofligament or tendon insertion repair.

UBM powder was produced as described previously [Freytes, D O et al,Biomaterials, 2004. 25(12): 2353-61]. A UBM gel digest was prepared bymixing one gram of lyophilized UBM powder with 100 mg of pepsin in 100mL of 0.01 M HCl under constant stirring for ˜48 hrs at roomtemperature. UBM gel polymerization was initiated by bringing the pH andthe ionic strength to physiological range using PBS at 37° C. Completepolymerization of the gel occurred within 30 min. The porous metalscaffolds were cleaned with acetone, methanol, and water, and passivatedwith 20-45% nitric acid.

The contact angles between the UBM gel digest (before [10 mg/ml] andafter physiologic activation [6 mg/ml]) and sheets of CP Ti or Ti 6Al 4Vwere measured. One ml of the digest was added to each surface and adigital photograph was taken for subsequent angle determination.

Two methods tested for ability to promote penetration of the polymerizedUBM gel into either CP titanium fiber mesh or CP titanium sinteredbeads. The entire surface of each porous metal scaffold was covered withactivated UBM gel for 5 minutes. For half of the scaffolds, the gel waspermitted to penetrate under static conditions, while in the other halfof the samples penetration took place using ultrasonication. Dye wasadded to visualize the gel.

To verify the presence of UBM gel within the porous titanium scaffoldsand to better understand the interaction between the UBM gel and themetal, specimens were prepared for environmental scanning electronmicroscopy (ESEM). Since the samples were able to be visualized withESEM while still in the hydrated condition, the interactions between thetitanium scaffold and the UBM gel were able to be determined withoutdisruption of the ECM by dehydration.

Both the UBM gel digest and the activated UBM gel wet the surface of theCP Ti and Ti 6Al 4V well (Table 4). Therefore, the porous metal shouldnot exclude the ECM. Based on these results, subsequent experimentsfocused on the activated gel. The UBM gel was able to penetrate half waythrough the thickness of each porous metal scaffold in the staticcondition. With the addition of ultrasonication, pores were infiltratedthrough the entire thickness of the scaffold (FIG. 17). Examination ofthe hybrid scaffolds with ESEM showed excellent penetration within andcoverage of the porous titanium (FIG. 18).

It is possible to create a hybrid ECM/porous metal scaffold using UBMgel and a porous titanium scaffold. Future studies will evaluate whetherthese scaffolds can support cell growth in vitro and promote connectivetissue ingrowth in vivo. The eventual goal of this effort is to developa scaffold that will promote ingrowth of soft tissue into the metal toserve as an insertion site for ligaments and tendons.

TABLE 4 Contact angle for UBM material on titanium alloys (Mean ± SD)Type of Metal UBM digest UBM gel CP Ti 46.8 ± 1.3 27.0 ± 4.0 Ti6Al4V38.2 ± 4.8 41.3 ± 1.6

1-96. (canceled)
 97. A composition comprising: (a) extracellular matrixderived from a mammalian tissue and (b) an acid protease.
 98. Thecomposition of claim 97, wherein the extracellular matrix is comminuted.99. The composition of claim 98, wherein the extracellular matrix islyophilized.
 100. The composition of claim 97, wherein the compositionis a liquid at a temperature of about 4° C. to 25° C. and a gel attemperature greater than 25° C.
 101. The composition of claim 97,wherein the mammalian tissue comprises cardiac tissue.
 102. Thecomposition of claim 97, wherein the extracellular matrix is digested.103. The composition of claim 102, wherein the composition has a pHbetween 7.2 and 7.8.
 104. The composition of claim 97, wherein theprotease is pepsin.
 105. The composition of claim 97, wherein thecomposition is a liquid.
 106. The composition of claim 105, wherein thecomposition transitions to a gel form at a physiological temperature.107. The composition of claim 105, wherein the extracellular matrix issuspended in the liquid.
 108. The composition of claim 105, wherein theextracellular matrix is solubilized in the liquid.
 109. The compositionof claim 97, wherein the composition is injectable through a needle.110. The composition of claim 97, wherein the composition is chemicallycross-linked.
 111. The composition of claim 97, wherein theextracellular matrix comprises collagen and glycosaminoglycan.
 112. Thecomposition of claim 97, wherein the composition further comprises abiocompatible material, a cell, a drug, a growth factor or anantibiotic.
 113. The composition of claim 97, wherein the extracellularmatrix is a non-dialyzed extracellular matrix.
 114. A method forcorrecting a disease-induced tissue defect in a subject in need ofrepair or augmentation of said tissue defect comprising administering tosaid subject the composition of claim
 97. 115. A liquid compositioncomprising: a) decellularized extracellular matrix derived frommammalian tissue; and b) water; wherein the liquid composition has adynamic viscosity that decreases with an increasing frequency.
 116. Theliquid composition of claim 115, wherein the liquid composition is ashear thinning liquid.
 117. The liquid composition of claim 115, whereinthe liquid composition further comprises a protease.
 118. The liquidcomposition of claim 117, wherein the protease is an acid protease. 119.The liquid composition of claim 117, wherein the protease is pepsin ortrypsin.
 120. The liquid composition of claim 115, wherein the liquidcomposition transitions to a gel form at a temperature that is higherthan 25° C.
 121. The liquid composition of claim 115, wherein the liquidcomposition transitions to a gel form at a physiological temperature.122. The liquid composition of claim 115, wherein the mammalian tissueis cardiac tissue.
 123. The liquid composition of claim 115, wherein thedecellularized extracellular matrix comprises collagen andglycosaminoglycan.
 124. The liquid composition of claim 115, whereinsaid liquid composition further comprises a biocompatible material, acell, a drug, a growth factor or an antibiotic.
 125. The liquidcomposition of claim 115, wherein the liquid composition is notdialyzed.
 126. A method for correcting a disease-induced tissue defectin a subject in need of repair or augmentation of said tissue defectcomprising administering to said subject the composition of claim 115.